Localization, mechanism and reduction of renal retention of technetium‐99m labeled epidermal growth factor receptor‐specific nanobody in mice

Abstract

Background: Nanobodies are single-domain antigen binding fragments derived from functional heavy-chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti-EGFR 99m Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of 99m Tc-7C12. Methods: First we compared the renal uptake of 99m Tc-7C12 in megalin-deficient and megalin-wild-type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of 99m Tc-7C12 was analyzed in CD-1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload R gelofusine coadministration (LysPreload R GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad R GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts. Results: Renal uptake of 99m Tc-7C12 was 44.22 W 3.46% lower in megalin-deficient compared with megalinwild-type mice. In CD-1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 W 2.99 and 36.22 W 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 W 2.09%. Gelofusine and lysine coadministration improved tumor uptake. Conclusion: Megalin contributes to the renal accumulation of 99m Tc-7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy.

A Fusion Protein of RGD4C and β-Lactamase Has a Favorable Targeting Effect in Its Use in Antibody Directed Enzyme Prodrug Therapy

“…The 99m Tc-RGD4CβL is rapidly cleared from the blood, mainly via the kidneys. This property agreed the typical behaviour of peptides and small proteins whose molecular weight is below the threshold that can be filtered by the glomerular membrane [19]. Meanwhile, 99m Tc-RGD4CβL was eliminated relatively slow in tumor compared with rapid clearance from blood, which means that the prodrug can be administrated when the fusion protein eliminated from normal tissues and the residual in tumor site can release the cephalosporin prodrug to kill tumor cells.…”

Targeted Radionuclide Therapy with A 177Lu-labeled Anti-HER2 Nanobody

“…Strategies to reduce the kidney uptake of radiolabeled protein- or peptide-based vectors have already been investigated intensively. For example, coinfusion with a mixture of 150 mg/kg Gelofusin and 1.2 g/kg L-lysine could reduce renal uptake of a 99m Tc-labeled anti-EGFR nanobody with 45 % 10. Gelofusin also reduced the kidney retention of radiolabeled octreotide analogs to a similar extent 29,30 and a combination of Gelofusin and L-lysine reduced kidney uptake even further 9,31.…”

“…Radiation nephropathy has indeed been observed in several patients after PRRT using radiolabeled somatostatin analogues 5-7. Recently, several countermeasures were described to reduce kidney retention of radiolabeled targeting probes 8-10, of which positively charged amino acid and/or Gelofusin coinfusions became standard practice in PRRT. Gelofusin consists of succinylated bovine gelatin molecules and is clinically used as a plasma expander.…”

Radiolabeled nanobodies as theranostic tools in targeted radionuclide therapy of cancer

“…Gainkam and coworkers showed that co-administering a mixture of 150 mg/kg Gelofusin and 1.2 g/kg l -lysine reduced renal uptake of a 99m Tc-labeled anti-EGFR nanobody with 45%, as the tumor-to-kidney ratio went up from 0.03 to 0.07 at 1.5 h p.i., in A431 tumor-xenografted mice [72]. Another approach to reduce renal retention of nanobodies consists of mutagenizing the protein sequence at sites that are responsible for interaction with the megalin/cubulin system.…”